PROJECT SUMMARY Sleep disorders affect over 10% of the population, and adequate sleep is essential for human health and cognitive function. Sleep is also apparently conserved across the animal kingdom. However, despite its importance and ubiquity, surprisingly little is known about the genetic, molecular, and neuronal mechanisms that govern sleep. Treatments for sleep disorders hinge on a better understanding of these mechanisms. Exogenous melatonin has long been recognized as a sleep-promoting molecule in humans, but endogenous melatonin has only recently been shown to be required for normal sleep levels. The latter studies were performed using zebrafish, a diurnal vertebrate whose brain resembles that of mammals and whose sleep is regulated by the same genes and neuronal populations that control sleep in mammals. Zebrafish larvae are highly amenable to high-throughput behavioral analysis and to genetic and neuronal manipulations. I plan to further exploit the tractability and convenience of zebrafish larvae to characterize the downstream signaling pathways and neuronal populations through which melatonin exerts its sleep-promoting effects. First, I will test the hypothesis that one or more melatonin receptor orthologs mediates the effects of melatonin on sleep by generating zebrafish containing mutations in each gene and assaying these mutants for sleep defects. Second, I will identify the neuronal populations that act downstream of melatonin by examining melatonin-induced changes in cfos expression and in GCaMP6s fluorescence, both indicators of neuronal activity, in populations of cells across the entire larval zebrafish brain. Third, I will test the hypothesis that melatonin promotes sleep by stimulating adenosine signaling, a pathway known to promote sleep in both zebrafish and mammals. The results of these experiments will help to confirm the role of endogenous melatonin as an important sleep regulator, and will identify genetic and neuronal substrates of melatonin-induced sleep. Because melatonin and adenosine are thought to mediate circadian and homeostatic sleep regulation, respectively, a possible interaction between these two molecules will lead to exciting new hypotheses about how homeostatic and circadian control of sleep are coordinated. The results of this study may eventually help guide the development and implementation of new therapies, and the refinement of existing therapies, for sleep and sleep-related disorders.